Virus-like particles (VLPs) are non-infectious, have repetitive surfaces that can display molecules with a high surface density, and have comparable cellular uptake and intracellular trafficking compared to natural virus. All of these functional attributes make them attractive as the assembly core for vaccines, diagnostics, and therapeutics. They can potentially serve as polyvalent scaffolds for the display of nucleic acids, proteins, and other chemical moieties. VLPs are particularly attractive as vaccines as they offer in vivo stability, trafficking to lymph nodes, and stimulation of B and T cell responses by the displayed epitopes. They can also be filled with cargo to serve as delivery vehicles.
Cell-free protein synthesis (CFPS) can be an effective method for producing VLPs, for example those comprising Hepatitis B core protein (HBc), MS2 bacteriophage coat protein, and Qβ bacteriophage coat protein, and the like. CFPS also provides a facile means for introducing non-natural amino acids (nnAAs) into proteins, which allows for the direct protein-protein coupling of antigens to VLPs using Cu(I)-catalyzed [3+2] cycloaddition click chemistry.
Among different types of VLPs, the HBc VLP is a flexible and promising model for knowledge-based display of foreign peptide sequences. The HBc particle was first reported as a promising VLP carrier in 1986. Being one of the first VLP candidates and the first icosahedral VLP carrier, HBc VLP has been well characterized and widely used as a carrier for over 100 different foreign sequences. The HBc capsid protein is 183 to 185 amino acids long. The arginine-rich C-terminus of HBc protein is dispensable for VLP assembly, so the HBc protein truncated at amino acid 149 is widely used. The truncated HBc (1-149) proteins can self-assemble into the particle with an average diameter of 28 to 30 nm and a dominant icosahedral symmetry of T=4.
However, in current applications of HBc VLPs, there is a serious problem. The VLP is not stable during click chemistry conjugations, and can disassemble after conjugation with functional molecules. Two truncated HBc monomers (16.7 kDa) form a dimer (33.5 kDa) by an intradimer C61-C61 disulfide bond. Then 120 dimers self-assemble into one VLP by hydrophobic interactions. Because the interdimer contacts are weak, conjugation of functional molecules onto the VLP surface can disturb the interactions between VLP dimers, resulting in VLP instability. The second problem is molecules with negative charges conjugate poorly to VLPs. At physiological pH, the surface of the HBc VLP is negatively charged. Because like charges repel, molecules with negative charges cannot get close to HBc VLP, and therefore the click chemistry conjugation cannot proceed effectively.
The present invention addresses these two problems, and provides stabilized HBc VLPs and a modified VLP surface.
Relevant Literature
Methods of introducing unnatural amino acids during CFPS are described in patent publication US 2010-0093024 A1. Methods of directly linking antigens and other polypeptides to a virus-like particle through unnatural amino acids are described in patent application US-2010-0168402-A1. Methods of encapsidating cargo into virus-like particles produced by CFPS are described in patent publication US-2010-0167981-A1. Each of these documents are herein specifically incorporated by reference.